Paracrine Influence Research Articles

Inhibition of angiogenesis by the secretome from iPSC-derived retinal ganglion cells with Leber's hereditary optic neuropathy-like phenotypes

The blood supply in the retina ensures photoreceptor function and maintains regular vision. Leber's hereditary optic neuropathy (LHON), caused by the mitochondrial DNA mutations that deteriorate complex I activity, is characterized by progressive vision loss. Although some reports indicated retinal vasculature abnormalities as one of the comorbidities in LHON, the paracrine influence of LHON-affected retinal ganglion cells (RGCs) on vascular endothelial cell physiology remains unclear. To address this, we established an in vitro model of mitochondrial complex I deficiency using induced pluripotent stem cell-derived RGCs (iPSC-RGCs) treated with a mitochondrial complex I inhibitor rotenone (Rot) to recapitulate LHON pathologies. The secretomes from Rot-treated iPSC-RGCs (Rot-iPSC-RGCs) were collected, and their treatment effect on human umbilical vein endothelial cells (HUVECs) was studied. Rot induced LHON-like characteristics in iPSC-RGCs, including decreased mitochondrial complex I activity and membrane potential, and increased mitochondrial reactive oxygen species (ROS) and apoptosis, leading to mitochondrial dysfunction. When HUVECs were exposed to conditioned media (CM) from Rot-iPSC-RGCs, the angiogenesis of HUVECs was suppressed compared to those treated with CM from control iPSC-RGCs (Ctrl-iPSC-RGCs). Angiogenesis-related proteins were altered in the secretomes from Rot-iPSC-RGC-derived CM, particularly angiopoietin, MMP-9, uPA, collagen XVIII, and VEGF were reduced. Notably, GeneMANIA analysis indicated that VEGFA emerged as the pivotal angiogenesis-related protein among the identified proteins secreted by health iPSC-RGCs but reduced in the secretomes from Rot-iPSC-RGCs. Quantitative real-time PCR and western blots confirmed the reduction of VEGFA at both transcription and translation levels, respectively. Our study reveals that Rot-iPSC-RGCs establish a microenvironment to diminish the angiogenic potential of vascular cells nearby, shedding light on the paracrine regulation of LHON-affected RGCs on retinal vasculature.

Abstract 7349: Ligand-receptor signaling analysis and tumor-immune microenvironment deconvolution reveal differential adipose-tumor crosstalk in lean and obese colon cancer patients

Abstract Obesity is a well-defined risk factor for colon cancer (CC). In the current study, we investigated the paracrine influences between tumors and adjacent adipose tissue to understand the interactions between adipose-tumor using transcriptomics data. Stage I-III newly diagnosed CC patients (n = 130) as part of the ColoCare Study at the Huntsman Cancer Institute, University of Utah, and University of Heidelberg, Germany were included in the current study. Fresh-frozen colon tumor tissues and adjacent adipose tissues naïve to neoadjuvant treatment were collected during surgery. RNA sequencing was done using the NovaSeq X. Patients were classified as having a BMI <30 kg∙m−2 (lean) or BMI ≥30 kg∙m−2 (obese). Tumor-adipose signaling was investigated separately for lean versus obese patients. Differentially expressed genes (DEGs, FDRq<0.05 and |log2FC|>2) between paired tumor-adipose samples were identified through DESeq2, respectively for lean and obese participants. DEGs were filtered to obtain exclusively up-regulated genes for 1) tumor tissue in the obese 2) tumor tissue in the lean 3) adipose tissue in the obese 4) adipose tissue in the lean. Ligand-receptor pairs for adipose-adipose signaling (A-A), tumor-tumor signaling (T-T), adipose-tumor signaling (A-T), and tumor-adipose signaling (T-A) in tissues from lean versus obese patients were determined by mapping the exclusively up-regulated genes in the FANTOM 5 database. Single sample Gene Set Enrichment Analysis (ssGSEA) was performed to score each sample based on signatures of interest, i.e. TIMEx immune cell types. The associations of signatures between tumor-adipose were assessed by evaluating Spearman’s correlation coefficients on the enrichment scores of each signature for paired tumor-adipose samples. From RNA-seq, there were 430 protein-coding DEGs (144 up-regulated in the tumor, 286 up-regulated in the adipose) identified exclusively in the obese and; 704 protein-coding DEGs (421 up-regulated in the tumor, 283 up-regulated in the adipose) identified exclusively in the lean patients. In the obese, we observed ligand-receptor-mediated T-T signaling through FGF5-FGFR3 and PYY-NPY4R; T-A signaling through GCG-GCGR; A-A signaling through ICAM1-IL2RA. In the lean, we found ligand-receptor-mediated T-T signaling through ADAM2-ITGA6 and LAMB3-ITGA6; T-A signaling through DLL3-NOTCH2; A-A signaling through DLK1-NOTCH2. In addition, ssGSEA analysis showed different association patterns for the lean versus obese regarding the TIMEx immune cell types. Our results suggest differential cross-talk between adipose and tumor tissue in lean vs. obese patients could influence the progression of CC. The next steps are to validate these interactions by using in vivo preclinical models to interrogate novel therapeutic targets for reducing the impact of obesity on CC. Citation Format: Tengda Lin, Victoria M. Bandera, Elaine M. Glenny, Caroline Himbert, Jennifer Ose, Christy Warby, Olena Aksonova, Alessandro Carpanese, Chris Stubben, David Nix, Kenneth M. Boucher, Peter Schirmacher, Ildiko Strehli, Jolanta Jedrzkiewicz, Courtney L. Scaife, Bartley Pickron, Alexander Brobeil, Martin Schneider, Christoph Kahlert, Erin M. Siegel, Adetunji T. Toriola, David Shibata, Christopher I. Li, Jane C. Figueiredo, Jatin Roper, Biljana Gigic, Stephen D. Hursting, Cornelia M. Ulrich, Aik Choon Tan. Ligand-receptor signaling analysis and tumor-immune microenvironment deconvolution reveal differential adipose-tumor crosstalk in lean and obese colon cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7349.

Assessing the functional potential of conditioned media derived from amniotic epithelial stem cells engineered on 3D biomimetic scaffolds: An in vitro model for tendon regeneration

Tendon diseases pose a significant challenge in regenerative medicine due to the limited healing capacity of this tissue. Successful tendon regeneration requires a combination of angiogenesis, immune response, and tenogenesis processes. An effective tendon engineering (TE) strategy must finely tune this systems’ interplay toward homeostasis.This study explores in vitro the paracrine influence of amniotic epithelial stem cells (AECs) engineered on a validated 3D electrospun PLGA scaffolds on HUVECs (angiogenesis), PBMCs/Jurkat (immune response), and AECs (tenogenic stem cell activation).The results revealed the role of scaffold's topology and topography in significantly modulating the paracrine profile of the cells. In detail, AECs basal release of bioactive molecules was boosted in the cells engineered on 3D scaffolds, in particular VEGF-D, b-FGF, RANTES, and PDGF-BB (p < 0.0001 vs. CMCTR). Moreover, biological tests demonstrated 3D scaffolds' proactive role in potentiating AECs' paracrine inhibition on PBMCs proliferation (CM3Dvs. CTR, p < 0.001) and LPS-mediated Jurkat activation with respect to controls (CM3D and CM2Dvs. CTR, p < 0.01 and p < 0.05, respectively), without exerting any in vitro pro-angiogenic role in promoting HUVECs proliferation and tubule formation. Teno-inductive paracrine ability of AECs engineered on 3D scaffolds was assessed on co-cultured ones, which formed tendon-like structures. These latter demonstrated an upregulation of tendon-related genes (SCX, THBS4, COL1, and TNMD) and the expression TNMD and COL1 proteins.Overall, this research underscores the pivotal role of the 3D topology and topography of PLGA tendon mimetic scaffolds in orchestrating effective tendon regeneration through modulating cell behavior and crosstalk between engineered stem cells and different subpopulations in the damaged tendon.

Fetoplacental vascular effects of maternal adrenergic antihypertensive and cardioprotective medications in pregnancy.

Maternal cardiovascular diseases, including hypertension and cardiac conditions, are associated with poor fetal outcomes. A range of adrenergic antihypertensive and cardioprotective medications are often prescribed to pregnant women to reduce major maternal complications during pregnancy. Although these treatments are not considered teratogenic, they may have detrimental effects on fetal growth and development, as they cross the fetoplacental barrier, and may contribute to placental vascular dysregulation. Medication risk assessment sheets do not include specific advice to clinicians and women regarding the safety of these therapies for use in pregnancy and the potential off-target effects of adrenergic medications on fetal growth have not been rigorously conducted. Little is known of their effects on the fetoplacental vasculature. There is also a dearth of knowledge on adrenergic receptor activation and signalling within the endothelium and vascular smooth muscle cells of the human placenta, a vital organ in the maintenance of adequate blood flow to satisfy fetal growth and development. The fetoplacental circulation, absent of sympathetic innervation, and unique in its reliance on endocrine, paracrine and autocrine influence in the regulation of vascular tone, appears vulnerable to dysregulation by adrenergic antihypertensive and cardioprotective medications compared with the adult peripheral circulation. This semi-systematic review focuses on fetoplacental vascular expression of adrenergic receptors, associated cell signalling mechanisms and predictive consequences of receptor activation/deactivation by antihypertensive and cardioprotective medications.

Studying Luminal, A and B subtypes of the breast cancer under paracrine secretion of fibroblasts

Introduction Understanding the role of the tumor microenvironment in specifying molecule markers of breast cancer subtypes could involve tumor microenvironment properties in subtyping and clinical assessment of breast cancer. Therefore, interconversion of luminal states and or their specific markers alteration under the control of TME particularly CAFs deserve to be more investigated. Methods To activate normal human fibroblasts, liquid overlay technique or nemesis was used and α-SMA expression, CAFs marker, in fibroblastic spheroids was measured by blotting. The luminal A, MCF-7, and luminal B, MDA-MB 361, cell lines are treated with normal and spheroidal/activated fibroblast conditioned medium for 48 hours. The morphological changes of luminal A and B cells were evaluated by invert microscopy and analyzed by the shape factor formula. Moreover, sensitivity changes to chemotherapeutic agents, Ki67 expression, and gene expression level including ER-related and proliferative genes were assessed respectively by MTT assay, Immunofluorescence, and real time qRT-PCR methods. Results Activated fibroblast, spheroid form, by LOT/nemosis expressed α-SMA marker two folds more than monolayer cultured fibroblasts. Our study underlined a significant increase in IC50 of both luminal A and B cell lines after being co-cultured with conditioned medium markedly in spheroidal conditioned medium. Morphological changes study by shape factor formula demonstrated more aggressiveness with gaining mesenchymal features both in luminal A and B subtypes by increasing exposure time. Changes in Ki67 expression level were observed after fibroblasts and spheroidal paracrine conditioning. Only this data supports the luminal interconversion by elevated Ki67 expression in luminal A and lowered Ki67 expression in luminal B. Gene expression results revealed that the MCF7 and MDA-MB cells as representatives luminal A and B subtypes in this study did not interchange their own specific gene pattern, however both luminal types in co-culture with condition medium increased the expression of anti-apoptotic Bcl2 gene. Conclusion Under the paracrine influence of fibroblast cells, both luminal A(MCF7) and B (MDA-MB) subtypes of breast cancer gained invasive, anti-apoptotic, and chemoresistance features which are mostly increased by activated fibroblast conditioned medium mimicking CAFs. There was no strong proof for interconversion of luminal A and B, with more similarities, subtypes of breast cancer.

Xenograft model of heterotopic transplantation of human ovarian cortical tissue and its clinical relevance.

Xenografts of human ovarian cortical tissue provide a tractable model of heterotopic autotransplantation that is used for fertility preservation in patients undergoing ablative chemo/radiotherapy. This study describes the behavior of hundreds of xenografts to establish a framework for the clinical function of ovarian cortex following autotransplantation over short- and long-term intervals. More than 200 live births have been achieved using autotransplantation of cryopreserved ovarian cortical fragments, yet challenges remain to be addressed. Ischemia of grafted tissue undermines viability and longevity, typically requiring transplantation of multiple cortical pieces; and the dynamics of recruitment within a graft and the influence of parameters like size and patient age at the time of cryopreservation are not well-defined. Here, we describe results from a series of experiments in which we xenografted frozen/thawed human ovarian tissue (n = 440) from 28 girls and women (age range 32 weeks gestational age to 46 years, median 24.3 ± 4.6). Xenografts were recovered across a broad range of intervals (1-52 weeks post-transplantation) and examined histologically to quantify follicle density and distribution. The number of antral follicles in xenografted cortical fragments correlated positively with the total follicle number and was significantly reduced with increased patient age. Within xenografts, follicles were distributed in focal clusters, similar to the native ovary, but the presence of a leading antral follicle coincided with increased proliferation of surrounding follicles. These results underscore the importance of transplanting ovarian tissue with a high density of follicles and elucidate a potential paracrine influence of leading antral follicles on neighboring follicles of earlier stages. This temporal framework for interpreting the kinetics of follicle growth/mobilization may be useful in setting expectations and guiding the parameters of clinical autotransplantation.

Endothelial cell‑derived connective tissue growth factor stimulates fibroblast differentiation into myofibroblasts through integrin αVβ3.

Connective tissue growth factor (CTGF) is expressed at high levels in blood vessels, where it functions as a regulator of a number of physiological processes, such as cell proliferation, angiogenesis and wound healing. In addition, CTGF has been reported to be involved in various pathological processes, such as tumor development and tissue fibrosis. However, one of the main roles of CTGF is to promote the differentiation of fibroblasts into myofibroblasts, a process that is involved in disease progression. Therefore, the present study aimed to investigate the possible mechanism by which pathological changes in the microvasculature can direct the activation of fibroblasts into myofibroblasts in the context of hypoxia/reoxygenation (H/R). Human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts were used in the present study. The expression levels of CTGF were determined by western blot analysis and reverse transcription-semi-quantitative PCR. To analyze the paracrine effect of HUVECs on fibroblasts, HUVECs were infected with CTGF-expressing adenovirus and then the culture supernatant of HUVECs was collected to treat fibroblasts. The formation of α-smooth muscle actin (α-SMA) stress fibers in fibroblasts were observed by immunofluorescence staining. It was found that H/R significantly increased CTGF expression in HUVECs. CTGF was also able to directly induce the differentiation of fibroblasts into myofibroblasts. In addition, the culture supernatant from CTGF-overexpressing HUVECs stimulated the formation of α-SMA stress fibers in fibroblasts, which was inhibited by treatment with a functional blocking antibody against integrin αVβ3 and to a lesser degree by a blocking antibody against α6 integrin. The mechanism of CTGF upregulation by H/R in HUVECs was then evaluated, where it was found that the CTGF protein was more stable in the H/R group compared with that in the normoxic control group. These findings suggest that CTGF expressed and secreted by vascular endothelial cells under ischemia/reperfusion conditions can exert a paracrine influence on neighboring fibroblasts, which may in turn promote myofibroblast-associated diseases. This association may hold potential as a therapeutic target.

Measurement of trace elements concentrations in human adipose tissue and links with phenotypic characteristics

Obesity is an international public health concern and it is related to various metabolic disorders such as type 2 diabetes mellitus and cardiovascular diseases [WHO, Obesity and overweight, 2021, online]. Some trace elements (i.e. Zn, Cd, Hg and Pb) have showed obesogen-like abilities [W. Shao, Environ Sci Pollut Res, 2017, 24, 11573–11581] but studies on chronic organs accumulation are limited. White adipose tissue (WAT), recognized as an endocrine and paracrine organ, stores energy and fat but its expansion causes obesity. Two main classes of WAT have been identified according to their location: subcutaneous adipose tissue (SAT) – including abdominal and gluteofemoral adipose tissues, which are highly sensitive to insulin- and visceral adipose tissue (VAT), sensitive to lipolysis and inflammation. A specific depot of VAT is the epicardial one, which exerts a direct vasocrine and paracrine influence on the heart. Although each of these adipose tissue depots shows functional specificities, very few epidemiological studies investigated the concentrations of trace elements in adipose tissues. Here we monitored trace element levels in four different adipose tissue deposits and explored their relation with different phenotypic data. Quantification of 16 trace elements (V, Cr, Mn, Co, Cu, Zn, As, Se, Mo, Ag, Cd, Sn, Sb, Pt, Hg, Pb) was performed by inductively coupled plasma - mass spectrometry (7700 Series, Agilent) in four deposits of human adipose tissue: visceral (VAT), epicardial (EAT), subcutaneous (SAT) and gluteofemoral (GAT). Samples of 103 persons, collected during autopsies in our center, were analyzed. Phenotypic data (age, sex, BMI), diabetes status and post-mortem interval were known. Multiple linear regression was used to look for association between phenotypic variables and trace elements concentrations. Cu, Zn, Se, Mn and Cd were detected in all adipose tissues samples; other trace elements were found only in some of the deposits. For most individuals, visceral adipose tissue samples showed higher trace element concentrations than subcutaneous ones. Interestingly, different trace elements presented a specific patterns of accumulation in the deposits. Adipose tissue did not appear to be strongly impacted by post-mortem redistribution. Only EAT showed significant difference ( P < 0.01) in Cd concentrations, depending on post-mortem interval (PMI): higher concentrations were measured when PMI > 48 h compared to PMI < 12 h. Most trace elements concentrations in adipose tissues showed a significant and negative association with BMI (for Co, Cu, Zn, Se, Mo in the four deposits and for V, Cr, Mn, Cd, Hg, Pb only in some of them). Association with age was significant and positive for As, Cd and Sn in some deposits and association with sex was significant only in few cases. Men presented significant higher arsenic levels than women in the four deposits and women showed significant higher concentrations of Cr (in EAT), Mn (in VAT), Se (in VAT), Mo (in VAT), Ag (in EAT), Cd (in VAT, EAT, and SAT) than men. Hypothesis of long-term accumulation of trace elements in adipose tissue is conceivable, yet it appeared to be element- and location-specific. Although the limited number of subjects included in this cohort was not enough to conclude on the significance of some outcomes, the tendencies observed raise new questions on the possible impact of trace elements on adipose tissue function. Therefore, further research is needed on this topic to corroborate the hypotheses raised in this study.

Single Cell Transcriptome Sequencing of Zebrafish Testis Revealed Novel Spermatogenesis Marker Genes and Stronger Leydig-Germ Cell Paracrine Interactions.

Spermatogenesis in testis is an important process for sexual reproduction, and worldwide about 10–15 percent of couples suffer from infertility. It is of importance to study spermatogenesis at single cell level in both of human and model organisms. Currently, single-cell RNA sequencing technologies (scRNA-seq) had been extensively applied to the study of cellular components and its gene regulations in the testes of different species, including human, monkey, mouse, and fly, but not in zebrafish. Zebrafish was a widely used model organism in biology and had been extensively used for the study of spermatogenesis in the previous studies. Therefore, it is also important to profile the transcriptome of zebrafish testis at single cell level. In this study, the transcriptomes of 14, 315 single cells from adult male zebrafish testes were profiled by scRNA-seq, and 10 cell populations were revealed, including Leydig cell, Sertoli cell, spermatogonia cell (SPG), spermatocyte, and spermatids. Notably, thousands of cell-type specific novel marker genes were identified, including sumo3b for SPG, krt18a.1 for Sertoli cells, larp1b and edrf1 for spermatids, which were also validated by RNA in situ hybridization experiments. Interestingly, through Ligand-Receptor (LR) analyses, zebrafish Leydig cells demonstrated stronger paracrine influence on germ cells than Sertoli cells. Overall, this study could be an important resource for the study of spermatogenesis in zebrafish and might also facilitate the study of the genes associated with human infertility through using zebrafish as a model organism.

Rostral ventrolateral medulla, retropontine region and autonomic regulations

The rostral half of the ventrolateral medulla (RVLM) and adjacent ventrolateral retropontine region (henceforth RVLMRP) have been divided into various sectors by neuroscientists interested in breathing or autonomic regulations. The RVLMRP regulates respiration, glycemia, vigilance and inflammation, in addition to blood pressure. It contains interoceptors that respond to acidification, hypoxia and intracranial pressure and its rostral end contains the retrotrapezoid nucleus (RTN) which is the main central respiratory chemoreceptor. Acid detection by the RTN is an intrinsic property of the principal neurons that is enhanced by paracrine influences from surrounding astrocytes and CO2-dependent vascular constriction. RTN mediates the hypercapnic ventilatory response via complex projections to the respiratory pattern generator (CPG). The RVLM contributes to autonomic response patterns via differential recruitment of several subtypes of adrenergic (C1) and non-adrenergic neurons that directly innervate sympathetic and parasympathetic preganglionic neurons. The RVLM also innervates many brainstem and hypothalamic nuclei that contribute, albeit less directly, to autonomic responses. All lower brainstem noradrenergic clusters including the locus coeruleus are among these targets. Sympathetic tone to the circulatory system is regulated by subsets of presympathetic RVLM neurons whose activity is continuously restrained by the baroreceptors and modulated by the respiratory CPG. The inhibitory input from baroreceptors and the excitatory input from the respiratory CPG originate from neurons located in or close to the rhythm generating region of the respiratory CPG (preBötzinger complex).

3-D vascularized breast cancer model to study the role of osteoblast in formation of a pre-metastatic niche

Breast cancer cells (BCCs) preferentially metastasize to bone. It is known that BCCs remotely primes the distant bone site prior to metastasis. However, the reciprocal influence of bone cells on the primary tumor is relatively overlooked. Here, to study the bone-tumor paracrine influence, a tri-cellular 3-D vascularized breast cancer tissue (VBCTs) model is engineered which comprised MDA-MB231, a triple-negative breast cancer cells (TNBC), fibroblasts, and endothelial cells. This is indirectly co-cultured with osteoblasts (OBs), thereby constituting a complex quad-cellular tumor progression model. VBCTs alone and in conjunction with OBs led to abnormal vasculature and reduced vessel density but enhanced VEGF production. A total of 1476 significantly upregulated and 775 downregulated genes are identified in the VBCTs exposed to OBs. HSP90N, CYCS, RPS27A, and EGFR are recognized as upregulated hub-genes. Kaplan Meier plot shows HSP90N to have a significant outcome in TNBC patient survivability. Furthermore, compared to cancer tissues without vessels, gene analysis recognized 1278 significantly upregulated and 566 downregulated genes in VBCTs. DKK1, CXCL13, C3 protein and BMP4 are identified to be downregulated hub genes in VBCTs. Together, a multi-cellular breast cancer model and culture protocols are established to study pre-metastatic events in the presence of OBs.

An Autonomous Cannabinoid System in Islets of Langerhans.

While endocannabinoids (ECs) and cannabis were primarily studied for their nervous system effects, it is now clear that ECs are also produced in the periphery where they regulate several physiological processes, including energy storage, glucose and lipid metabolism, insulin secretion and synthesis, and hepatocyte function. Within islet of Langerhans there is an autonomous EC system (ECS). Beta (β)-cells contain all the enzymes necessary for EC synthesis and degradation; ECs are generated in response to cellular depolarization; their paracrine influence on β-cells is mostly through the cannabinoid 1 receptor (CB1R) that is present on all β-cells; they modulate basal and glucose- and incretin-induced insulin secretion, and β-cell responses to various stressors. Furthermore, there is now accumulating evidence from preclinical studies that the autonomous islet ECS is a key player in obesity-induced inflammation in islets, and β-cell damage and apoptosis from many causes can be mitigated by CB1R blockers. We will thoroughly review the literature relevant to the effects of ECs and their receptors on β-cells and the other cell types within islets. Therapeutic potential of agents targeting EC/CB1R and CB2R is highly relevant because the receptors belong to the druggable G protein-coupled receptor superfamily. Present research in the ECS must be considered preliminary, especially with regards to human islet physiology, and further research is needed in order to translate basic cellular findings into clinical practice and the use of safe, clinically approved CBR modulators with and without glucose lowering combinations presently in therapeutic use for diabetes and obesity needs to be studied.

KATP channel blockers control glucagon secretion by distinct mechanisms: A direct stimulation of α-cells involving a [Ca2+]c rise and an indirect inhibition mediated by somatostatin

ObjectiveGlucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from β- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. MethodsUsing a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. ResultsApplication of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in β-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in β-cells. ConclusionsSulfonylureas exert two opposite actions on α-cells: a direct stimulation as in β-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than β-cells.

The potential role of multifunctional human amniotic epithelial cells in pancreatic islet transplantation.

Pancreatic islet cell transplantation has proven efficacy as a treatment for type 1 diabetes mellitus, chiefly in individuals who are refractory to conventional insulin replacement therapy. At present its clinical use is restricted, firstly by the limited access to suitable donor organs but also due to factors associated with the current clinical transplant procedure which inadvertently impair the long-term functionality of the islet graft. Of note, the physical, biochemical, inflammatory, and immunological stresses to which islets are subjected, either during pretransplant processing or following implantation are detrimental to their sustained viability, necessitating repeated islet infusions to attain adequate glucose control. Progressive decline in functional beta (β)-cell mass leads to graft failure and the eventual re-instatement of exogenous insulin treatment. Strategies which protect and/or preserve optimal islet function in the peri-transplant period would improve clinical outcomes. Human amniotic epithelial cells (HAEC) exhibit both pluripotency and immune-privilege and are ideally suited for use in replacement and regenerative therapies. The HAEC secretome exhibits trophic, anti-inflammatory, and immunomodulatory properties of relevance to islet graft survival. Facilitated by β-cell supportive 3D cell culture systems, HAEC may be integrated with islets bringing them into close spatial arrangement where they may exert paracrine influences that support β-cell function, reduce hypoxia-induced islet injury, and alter isletalloreactivity. The present review details the potential of multifunctional HAEC in the context of islet transplantation, with a focus on the innate capabilities that may counter adverse events associated with the current clinical transplant protocol to achieve long-term islet graft function.

Adrenal cortex renewal in health and disease.

Resident progenitor and/or stem cell populations in the adult adrenal cortex enable cortical cells to undergo homeostatic renewal and regeneration after injury. Renewal occurs predominantly in the outer layers of the adrenal gland but newly formed cells undergo centripetal migration, differentiation and lineage conversion in the process of forming the different functional steroidogenic zones. Over the past 10 years, advances in the genetic characterization of adrenal diseases and studies of mouse models with altered adrenal phenotypes have helped to elucidate the molecular pathways that regulate adrenal tissue renewal, several of which are fine-tuned via complex paracrine and endocrine influences. Moreover, the adrenal gland is a sexually dimorphic organ, and testicular androgens have inhibitory effects on cell proliferation and progenitor cell recruitment in the adrenal cortex. This Review integrates these advances, including the emerging role of sex hormones, into existing knowledge on adrenocortical cell renewal. An in-depth understanding of these mechanisms is expected to contribute to the development of novel therapies for severe endocrine diseases, for which current treatments are unsatisfactory.

The effects of cardiac stretch on atrial fibroblasts: analysis of the evidence and potential role in atrial fibrillation.

Atrial fibrillation (AF) is an important clinical problem. Chronic pressure/volume overload of the atria promotes AF, particularly via enhanced extracellular matrix (ECM) accumulation manifested as tissue fibrosis. Loading of cardiac cells causes cell stretch that is generally considered to promote fibrosis by directly activating fibroblasts, the key cell type responsible for ECM production. The primary purpose of this article is to review the evidence regarding direct effects of stretch on cardiac fibroblasts, specifically: (i) the similarities and differences among studies in observed effects of stretch on cardiac fibroblast function; (ii) the signalling pathways implicated; and (iii) the factors that affect stretch-related phenotypes. Our review summarizes the most important findings and limitations in this area and gives an overview of clinical data and animal models related to cardiac stretch, with particular emphasis on the atria. We suggest that the evidence regarding direct fibroblast activation by stretch is weak and inconsistent, in part because of variability among studies in key experimental conditions that govern the results. Further work is needed to clarify whether, in fact, stretch induces direct activation of cardiac fibroblasts and if so, to elucidate the determining factors to ensure reproducible results. If mechanical load on fibroblasts proves not to be clearly profibrotic by direct actions, other mechanisms like paracrine influences, the effects of systemic mediators and/or the direct consequences of myocardial injury or death, might account for the link between cardiac stretch and fibrosis. Clarity in this area is needed to improve our understanding of AF pathophysiology and assist in therapeutic development.

Abstract 14765: Exosome Shuttled VEGFR Promotes Angiogenesis in Peripheral Vascular Disease

Introduction: Counteracting the pandemic of peripheral vascular disease necessitates development of proangiogenic treatments. As stem cell benefit has increasingly been associated with paracrine influence on diseased tissue, a purified extracellular vesicle population (PEP) was established to promote tissue regeneration and neovascularization. The mechanistic basis for PEP-induced proangiogenic effects was here probed and validated in two separate in vivo models of vascular ischemia. Hypothesis: PEP drives pro-angiogenic effects though donation of VEGFR. Methods: Nanosight and immunoblotting were utilized to characterize PEP purity and identity. OMICs profiling identified VEGFR as highly prioritized, with downstream signaling was tested in human umbilical vascular endothelial cells using Matrigel assay, qPCR and immunoblotting. In vivo, blood perfusion (Fluorescent imaging - SPY) was quantified in a rodent model of hind-limb ischemia and in a rabbit model of critical ischemic ear, each treated with sham or PEP. Rabbit tissues were next examined by whole transcriptome analysis to assess PEP-induced proangiogenic signaling. Immunohistochemical staining with smooth muscle actin and von Willebrand factor probed de novo blood vessel formation. Results: PEP was characterized by size (mean: 128.6 +/-1 nm) and VEGFR expression. VEGFR knock-down diminished endothelial cell angiogenic activity in vitro, rescued with PEP (5.3x1010+/-109 vesicles/ml) was sufficient enhance total and phosphor-VEGFR expression, along with amplification of downstream VEGFR targets (MAPK, AKT, CXCL8, NOTCH4, MMP9) and enhanced tube formation. In both hind-limb (n=5) and critical ear (n=4) ischemia models, local delivery of PEP to ischemic tissue resulted in restoration of blood perfusion and prioritized proangiogenic signaling in tissue transcriptome pathway analysis, further confirmed by immunohistochemistry. Conclusions: Taken together, our study establishes a new regenerative paradigm documenting exosome-based delivery of VEGFR as a key driver for angiogenesis. This PEP-based platform broadens potential options available for treatment of peripheral vascular disease and may have therapeutic implications in restoration of microvasculature.

Stromal-AR influences the growth of epithelial cells in the development of benign prostate hyperplasia.

Activation of epithelial-AR signaling is identified as the major cause of hyperproliferation of the cells during benign and malignant prostate conditions. However, the contribution of stromal-AR is also precarious due to its secretory actions that contribute to the progression of benign and malignant tumors. The present study was aimed to understand the influence of stromal-AR mediated actions on epithelial cells during BPH condition. The secretome (conditioned media-CM) was collected from AR agonist (testosterone-propionate-TP) and antagonist (Nilutamide-Nil) treated BPH patient-derived stromal cells and exposed to BPH epithelial cells. Epithelial cells exhibited increased cell proliferation with the treatment of CM derived from TP-treated stromal cells (TP-CM) but did not support the clonogenic growth of BPH epithelial cells. However, CM derived from Nil-treated stromal cells (Nil-CM) depicted delayed and aggressive BPH epithelial cell proliferation with increased clonogenicity of BPH epithelial cells. Further, decreased AR levels with increased cMyc transcripts and pAkt levels also validated the clonogenic transformation under the paracrine influence of inhibition of stromal-AR. Moreover, the CM of stromal-AR activation imparted positive regulation of basal/progenitor pool through LGR4, β-Catenin, and ΔNP63α expression. Hence, the present study highlighted the restricted disease progression and retains the basal/progenitor state of BPH epithelial cells through the activation of stromal-AR. On the contrary, AR-independent aggressive BPH epithelial cell growth due to paracrine action of loss stromal-AR directs us to reform AR pertaining treatment regimes for better clinical outcomes.

Spermatogonial Stem Cells in Fish: Characterization, Isolation, Enrichment, and Recent Advances of In Vitro Culture Systems.

Spermatogenesis is a continuous and dynamic developmental process, in which a single diploid spermatogonial stem cell (SSC) proliferates and differentiates to form a mature spermatozoon. Herein, we summarize the accumulated knowledge of SSCs and their distribution in the testes of teleosts. We also reviewed the primary endocrine and paracrine influence on spermatogonium self-renewal vs. differentiation in fish. To provide insight into techniques and research related to SSCs, we review available protocols and advances in enriching undifferentiated spermatogonia based on their unique physiochemical and biochemical properties, such as size, density, and differential expression of specific surface markers. We summarize in vitro germ cell culture conditions developed to maintain proliferation and survival of spermatogonia in selected fish species. In traditional culture systems, sera and feeder cells were considered to be essential for SSC self-renewal, in contrast to recently developed systems with well-defined media and growth factors to induce either SSC self-renewal or differentiation in long-term cultures. The establishment of a germ cell culture contributes to efficient SSC propagation in rare, endangered, or commercially cultured fish species for use in biotechnological manipulation, such as cryopreservation and transplantation. Finally, we discuss organ culture and three-dimensional models for in vitro investigation of fish spermatogenesis.

Impaired Microvascular Endothelial Function in Preeclampsia

RationalWomen with preeclampsia (PE) have compromised endothelial function (assessed by brachial artery flow‐mediated dilation) compared to normal women. Placental and uterine spiral artery endothelial dysfunction is seen in PE pregnancies. Human studies remain incomplete, as most approaches have been unable to eliminate neural, humoral, paracrine influences on vessel response. Direct evaluation of the placental arterioles will allow a more direct analysis of vasodilatory responses as related to the endothelial dysfunction in PE.ObjectiveTo study placental endothelial function in preeclamptic (PE) and normal placental vessels.MethodsTo examine the impact of PE on the microvascular function, isolated vessels from placentas of healthy and PE pregnancies were used for videomicroscopy. Endothelial‐dependent dilation to flow (FMD), acetylcholine (ACh) and smooth muscle‐dependent dilation to papaverine were evaluated. Data are presented as a percentage of maximum diameter and were analyzed with 2‐way ANOVA RM with statistical significance set at *p&lt;0.05; data are expressed as mean ± SE.ResultsVessels from PE patients resulted in impairment of FMD (control 71.% ±6.6%, PE 31.6%±10.7%*) and dilation to Ach (control 63.6%±9.4, PE 32.7±8.83%*). Smooth muscle‐dependent dilation to papaverine was unchanged between the two groups (Figure ).ConclusionPreliminary studies show impaired endothelial function in placental vessels of subjects with PE, compared to healthy controls, while smooth muscle function was not affected. Understanding the underlying mechanism(s) of vasodilation in placental vasculature will further the understanding the pathophysiology of PE.Support or Funding InformationThe project described was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, Award NumberUL1TR001436. The content is solely the responsibility of the author(s) and does not necessarily represent the official views of the NIH. This project is funded by the Research and Education Program Fund, a component of the Advancing a Healthier Wisconsin Endowment at the Medical College of Wisconsin.Vessels from PE patients resulted in impairment of FMD and dilation to ACh. Smooth muscle‐dependent dilation to papaverine was unchanged between the two groups. Statistical significance denoted with *Figure 1